1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it relates to the use of mass tag complexes to permit simultaneously obtaining of gene expression information on a plurality of targets. In addition, it permits one to perform spatial profiling, i.e., securing information on both expression level and cellular position.
2. Description of Related Art
Localization of specific messenger RNA (mRNA) and protein molecules within cells and tissues can provide important information on differential expression and help elucidate mechanisms of pathophysiologic changes. The most widely used techniques for assessing the cellular and tissue distribution of protein and mRNA are immunohistochemistry and in situ hybridization, respectively. In the clinical setting, immunohistochemistry is an established technique in modern cancer biology and oncology and many diagnoses are based on its findings. Since these techniques are used with sectioned tissues, the spatial and cellular resolution that is present in the whole organ or tumor is maintained. Although this allows a level of cellular resolution that is not possible with methodologies that require cell disruption or homogenization, a major limitation is that the number of transcripts that can be simultaneously detected is small. Generally, a single transcript of interest is probed in a tissue section with multiple, adjacent sections being used to detect larger numbers of transcripts in parallel assays.
In contrast, the wealth of genomic information from the genome sequencing projects combined with the development of high-density profiling technologies such as DNA microarrays now allows simultaneous profiling of tens of thousands of transcripts. The efficiency in the number of transcripts that can be simultaneously profiled is unparalleled compared to any other technique, however there are other limitations associated with microarray technologies. DNA microarrays are not suitable for in-situ assays and therefore do not allow spatial and cellular orientations of the profiled transcripts to be observed. Furthermore, the amount of RNA material that is required for a single microarray assay is quite large compared to the amount of RNA present in a single cell. Methods for isolating single cells or small groups of cells using laser capture microdissection followed by RNA amplification techniques have helped with this limitation, but efficiency is severely impacted and the use of microarrays to profile a significant number of individual cells or small groups of cells becomes impractical.
Recently, several reports have described the use of tag molecules to specifically label a population of proteins to allow a comparative analysis of two complex protein mixtures (Zhou et al., 2002; Han et al., 2001; Gygi et al., 1999). These tag molecules are identical in chemical structure, but differ in total mass. A “heavy” version of the tag contains deuterium, while the “light” version contains hydrogen, providing a difference in total mass based on the number of deuterium versus hydrogen atoms present. The remaining structure contains a reactive group to facilitate binding to proteins and an affinity tag, such as biotin. Together, these tags are referred to as isotope-coded affinity tags (ICAT). By labeling two complex protein mixtures isolated from two cell states with a heavy and light ICAT tag, respectively, the states can be differentially analyzed. Following labeling, the mixtures are combined, fractionated and analyzed by liquid chromatography-mass spectrometry (LC-MS). The same protein from each population can be identified, and a relative ratio between the same protein from the different cell states established based on the presence of the heavy or light affinity tag. A variation on this theme was recently described that adopts that ICAT method to the solid phase to increase the efficiency and reproducibility in the automation of the process. A similar isotope tag is coupled to a solid bead by a photocleavable linkage, which provides an efficient mechanism for the purification of the captured proteins or peptides followed by photocleavage away from the beads and analysis by LC-MS (Zhou et al., 2002).
Thus, there remains a need to develop technologies that allow the efficient and spatial profiling of a moderate number of genes from intact tissue specimens.